The human ARC (activator-recruited cofactor) complex has recently been discovered as a target and co-activator of a number of transcriptional activators that control the expression of important genes. Initially, ARC was found to be targeted by the transcriptional activators SREBP-1a, VP-16 and NF-KB, but more recent information has revealed that ARC also interacts with other transcriptional activators, such as [unreadable]-catenin, members of the Smad family and p53. Subsequently, the ARC coactivator complex was recognized as the human homolog of the yeast Mediator. It mediates recruitment of RNA polymerase II (Pol-II) to the promoter regions of specific genes by binding, on the one hand, to transactivation domains of DNA-bound activators, and on the other hand, engaging the C-terminal domain of the large subunit of Pol-II. The goal of the proposed research is to study interactions of viral and cellular transcriptional activators with two components of ARC, ARC92 and ARC105. These two components are the primary targets of prominent transcriptional activators that play pivotal roles in a wide variety of biological phenomena ranging from inflammation, apoptosis and lipid biosynthesis, to cancer and viral infection. We propose to use structural and biochemical methods for elucidating the molecular mechanisms of coactivator-activator recruitment. We will address questions of specificity and promiscuity of ARC interactions. The planned research will pursue three specific aims: Aim 1. Structural and functional characterization the SREBP interaction with ARC105 using NMR spectroscopy, cell biology approaches and small molecule inhibitors of their interaction. Aim 2. Determine the structure of the NF-KB/ARC105 complex and characterize its significance with biochemical methods and search for small-molecule inhibitors to test the significance of the interaction. Aim 3. Solve the structure of the ARC92 complex with the VP16 transactivation domain. Identify the minimal ARC92-binding domain of [unreadable]-catenin and solve the structure of the activator/co-activator complex. Search for small-molecule inhibitors of the interaction of ARC92 with the transactivation domains of VP16 and [unreadable]-catenin and use them to elucidate the significance of the interactions in in-vitro and in-vivo experiments.